1. Field of the Invention
The present invention relates generally to the field of molecular biology and immunology. More particularly, it concerns methods for high-throughput isolation cDNAs encoding immune cell receptors and antibodies.
2. Description of Related Art
There is a need to identify the expression of two or more transcripts from individual B cells expressing antigen-specific antibodies at high throughput (Bardelli et al. 2013, Love et al. 2006, Mazutis et al. 2013, Smith et al. 2009, Ogunniyi et al. 2009). In particular, for numerous biotechnology and medical applications it is important to identify and sequence the gene pairs encoding the two chains comprising adaptive immune receptors from individual cells at a very high throughput in order to accurately determine the complete repertoires of immune receptors expressed in patients or in laboratory animals. Immune receptors expressed by B lymphocytes are encoded by the VH and VL antibody genes. Humans have many tens of thousands or millions of distinct B cells. High-throughput DNA sequencing technologies have been used to determine repertoires of VH or VL chains of relevance to particular disease states or, more generally, to study the function of the adaptive immune system (Wu et al., 2011). Immunology researchers have an especially great need for high throughput analysis of multiple transcripts at once (DeKosky et al. 2013, Georgiou et al. 2014)
Currently available methods for immune repertoire sequencing involve mRNA isolation from a cell population of interest, e.g., memory B-cells or plasma cells from bone marrow, followed by RT-PCR in bulk to synthesize cDNA for high-throughput DNA sequencing (Reddy et al., 2010; Krause et al., 2011). However, heavy and light antibody chains (or α and β T-cell receptors) are encoded on separate mRNA strands and must be sequenced separately. Of particular interest is the determination of the sequences of VH and VL genes encoded by single B cells that express antibodies specific to a desired antigen. Without multiple-transcript analysis at the single-cell level to collect heavy and light chain pairing data from B cells expressing antigen-specific antibodies, the full adaptive immune receptor, which includes both chains, cannot be sequenced or reconstructed and expressed for further study.